Structure and dynamics of a functional cavity in the HIV-1 Envelope, and its role in conformational changes required for infection Channels and cavities in protein structures often play an important role in modulating structure, function and dynamics. The HIV-1 Envelope (Env) glycoprotein, a conformational machine that utilizes receptor binding mediated conformational changes to effect virus and host cell membrane fusion, is riddled with cavities and channels. A highly conserved cavity ? called the Phe43 cavity - engages the Phe43 residue of receptor CD4 and is critical for CD4-induced Env conformational transitions. The Phe43 cavity is also the binding site of broad and potent antibodies, drugs and peptide inhibitors of HIV-1 entry. The CD4-mimetic miniprotein M48U1, effects broad and potent HIV-1 neutralization, binds HIV-1 gp120 with pM affinity, showed efficacy as a vaginal microbicide in animal models, and more recently, was shown to synergize with the drug Tenofovir to inhibit HIV infection in activated PBMCs and human cervicovaginal histocultures. M48U1 inserts a methoxy cyclohexyl moiety to fill the Phe43 cavity. M48U1 and its analogs are the only class of ligands that reach deep into the Phe43 cavity, a property that makes them valuable tools for probing the structure of a deep, otherwise not easily accessible cavity.We have previously utilized M48U1 and its analogs to probe the structure of the Phe- 43 cavity in gp120 monomers. High resolution structures of M48U1 bound to HIV-1 Env gp120 revealed ligand flexibility, and the ability to fit within and adapt to the Phe43 cavities of diverse HIV-1 isolates with minimal perturbations of the cavity structure are key determinants of activity. In this grant we propose to explore the structure of the Phe43 cavity in the closed HIV-1 Env trimer using M48U1 as a molecular probe. The innovation in this grant derives from our use of M48U1 and its analogs as molecular probes of the Phe43 cavity, from the advances in cryo-EM technology that include improved specimen vitrification methods, improved microscope hardware, automated methods for high-throughput data collection, and advanced algorithms for data processing. These advances have recently allowed us to establish a rapid pipeline for determining high resolution structures of HIV-1 Env complexes. The scientific premise of this grant is that the Phe43 cavity is a critical functional component in HIV-1 Env, central to the activity of the CD4 receptor and many broad and potent antibodies and drugs. M48U1 is an exceptionally effective HIV-1 entry inhibitor, and one of the only ligands that fills the entire Phe-43 cavity. Exploring the structures of HIV-1 Env when bound to M48U1 will provide insights into the structure and the conformational constraints on the cavity in the closed state of the Env.